Measurement method, measurement device, program, and storage medium

Abstract

This invention provides a measurement method that enables the evaluation of the detection limit of a single sample in a measuring device. [Solution] The measurement method involves using multiple solutions in which multiple fine particles (2001) labeled with a fluorescent dye (2002) and emitting reference light are dispersed, measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the multiple fine particles; obtaining the proportion (detection rate r) of particles among the multiple fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected for each of the multiple solutions; and determining the minimum number of fluorescent dyes detectable by the measuring device (1000) (detection limit x) based on the proportions in each of the multiple solutions. min The process includes the step of obtaining ).

Description

Technical Field

[0001] The present invention relates to a measurement method, a measurement device, a program, and a storage medium.

Background Art

[0002] Development of measurement techniques for observing minute specimens such as viruses and extracellular vesicles at a single level and analyzing proteins expressed in individual specimens has been underway. In Patent Document 1 and Non-Patent Document 1, a method for measuring a single extracellular vesicle is disclosed by fixing an extracellular vesicle on a plasmon substrate using an affinity ligand that selectively binds to the extracellular vesicle and measuring fluorescence amplified by surface plasmon resonance.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] It is desirable to evaluate the detection limit of a single specimen in a measurement device.

Means for Solving the Problems

[0006] One aspect of the present invention is a measurement method comprising the steps of: measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the plurality of fine particles, using a plurality of solutions in which a plurality of fine particles labeled with a fluorescent dye and emitting reference light are dispersed; obtaining the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected for each of the plurality of solutions; and obtaining the minimum number of fluorescent dyes detectable by a measuring device based on the proportions for each of the plurality of solutions. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a measurement method that can evaluate the detection limit of a single sample in a measuring device. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram of the measuring device in Example 1. [Figure 2] This is a schematic diagram of the calibration particles in each example. [Figure 3] This is a schematic diagram showing the method for calculating the detection rate of calibration particles in each example. [Figure 4] This is a schematic diagram showing the method for calculating the detection limit in each example. [Figure 5] This flowchart shows the measurement methods for each embodiment. [Figure 6] This is a schematic diagram showing a measurement method as a modified example of each embodiment. [Figure 7] This figure shows the evaluation results in Example 1. [Figure 8] This is a schematic diagram of the measuring device in Example 2. [Modes for carrying out the invention]

[0009] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[0010] Figure 1 is a schematic diagram showing the configuration of the fluorescence measuring device (measuring device) 1000 in each embodiment. The fluorescence measuring device 1000 has a microscope unit (detection means) 1100, an illumination unit (illumination means) 1200, and a control unit 1300. The microscope unit 1100 and the illumination unit 1200 constitute the measuring means. As described later, the measuring means uses multiple solutions in which multiple fine particles labeled with an evaluation fluorescent dye and emitting reference light are dispersed, and measures the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the multiple fine particles. The fluorescence measuring device 1000 irradiates the sample 1500, which is placed on a substrate 1400 (chemically bonded to the substrate), with illumination light and detects (measures) the fluorescence emitted from the sample 1500.

[0011] The microscope unit 1100 comprises an optical system (measuring optical system) having an objective lens 1101 and an imaging lens 1102, and an image sensor 1103 such as a CMOS sensor. The magnified image of the sample 1500 formed by the objective lens 1101 and the imaging lens 1102 is imaged by the image sensor 1103. To obtain images at different magnifications, the objective lens 1101 may be mounted on a revolving nosepiece capable of accommodating multiple objective lenses 1101.

[0012] The illumination unit 1200 comprises a light source 1201 and an illumination optical system. The illumination optical system includes a collimator lens 1202, a focusing lens 1203, and a filter cube 1204.

[0013] In the configuration shown in Figure 1, the objective lens 1101 is shared between the microscope unit 1100 and the illumination unit 1200, and the illumination unit 1200 illuminates the sample 1500 with a configuration that includes the objective lens 1101.

[0014] The light source 1201 is an LED (light-emitting diode) or a laser light source, but is not limited to these. The light source 1201 may be configured, for example, by combining a light source with a broad wavelength range, such as a halogen lamp or a white LED, with an appropriate bandpass filter. The illumination unit 1200 constitutes a Köhler illumination system with a collimator lens 1202, a focusing lens 1203, and an objective lens 1101.

[0015] The filter cube 1204 has wavelength characteristics that reflect illumination light and transmit fluorescence emitted from the sample 1500. A filter cube with such wavelength characteristics can be constructed, for example, by combining a bandpass filter that transmits only illumination light, a dichroic mirror that reflects only illumination light and transmits fluorescence from the sample, and a bandpass filter that transmits only fluorescence. For a simpler configuration, the filter cube 1204 may consist of a combination of one bandpass filter and a dichroic mirror, or a configuration consisting only of a dichroic mirror. Alternatively, multiple filter cubes may be prepared and switched according to the target fluorescent dye. In this case, to facilitate switching, the filter cube to be used from among the multiple filter cubes may be mounted on a selectable filter wheel.

[0016] The control unit 1300 is equipped with a dedicated computer or personal computer and controls the lighting of the light source of the illumination unit 1200, the driving of a drive mechanism (not shown), and the image acquisition of the microscope unit 1100 according to a program. Specifically, the control unit 1300 communicates with the illumination unit 1200 to switch the illumination wavelength and the filter cube 1204, and also communicates with the microscope unit 1100 to acquire fluorescence images at each wavelength. Furthermore, the control unit 1300 may perform detection limit estimation calculations, which will be described later.

[0017] The control unit 1300 and each component may be directly connected by cables or the like, or they may be connected using a short-range communication system. In addition to controlling the microscope unit 1100 and the illumination unit 1200, the control unit 1300 may also have functions such as image retention, image-based calculations, and image display. These functions may be performed by another device via a network. By analyzing the acquired images, information such as proteins and RNA contained in the sample 1500 can be obtained.

[0018] The control unit 1300 has a first acquisition means 1301 and a second acquisition means. The first acquisition means 1301 determines, for each of the plurality of solutions, the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected (detection rate r). i The second acquisition means 1302 obtains the minimum number of fluorescent dyes detectable by the fluorescence measuring device 1000 (detection limit x) based on the proportion of each of the multiple solutions. min ) obtain.

[0019] The substrate 1400 is an element for fixing the sample 1500 to its surface for microscopic observation. It is a glass substrate, a substrate coated with a dielectric or metal on its surface, or a plasmon substrate that induces plasmon resonance due to the microstructure of the metal, but is not limited to these. The substrate 1400 may also have a ligand (a substance that specifically binds to a particular receptor) on its surface that selectively binds the sample 1500 to the substrate.

[0020] Sample 1500 is the object to be measured, and there are various types depending on the purpose of the measurement. When performing evaluation and calibration of the instrument, the fluorescent particles used for calibration, as described later, become Sample 1500. After calibration, for example, extracellular vesicles (such as exosomes) derived from biological tissue, viruses, liposomes, etc. become Sample 1500. In each example, the fluorescent particles used as Sample 1500 during calibration are referred to as calibration particles, and the Sample 1500 used for measurement after calibration is referred to as the specimen.

[0021] A fluorescent dye is bound to the sample via an antibody corresponding to the target protein. When a fluorescence image of the sample is acquired by the fluorescence analyzer 1000, multiple emission points are observed in the image if the sample contains the target protein. Since each emission point is caused by fluorescence emitted from individual samples containing the target protein, the amount of sample containing the target protein can be evaluated by assessing the number of emission points (detection count) in the image.

[0022] Such bright spots cannot be detected by the fluorescence analyzer 1000 unless a certain number of fluorescent dyes are bound to the sample. Therefore, it is important to evaluate the minimum number of fluorescent dyes per sample required for detection by the fluorescence analyzer 1000, that is, the detection limit in relation to the number of fluorescent dyes. One method for evaluating the detection limit of the fluorescence analyzer 1000 in relation to the number of dyes is to use calibration particles whose number of bound fluorescent dyes has been determined in advance, and then evaluate whether or not the particles can be detected.

[0023] Because fluorescence emitted from minute samples of approximately tens to hundreds of nanometers in size, such as extracellular vesicles and viruses, is weak, highly sensitive fluorescence measurement techniques are necessary. To improve sensitivity, methods such as fixing the sample on a plasmon substrate or a metal-coated substrate, as described in Patent Document 1, and observing it, can be considered. However, the intensity of emission from such substrates varies depending on the size of the sample, the refractive index, and the method of dye binding. In other words, the detection limit may change depending on the sample.

[0024] Therefore, when performing calibration, it is desirable to use fluorescent particles that simulate the size, refractive index, and dye binding method of the sample being measured. In this case, calibration particles need to be approximately tens to hundreds of nanometers in size, and even these calibration particles emit weak fluorescence. As a result, it is not possible to accurately evaluate the detection limit of the fluorescence measuring device 1000.

[0025] To address these challenges, each embodiment uses calibration particles in which a fluorescent dye for evaluation is bound to particles of a similar size to the sample, thereby emitting sufficiently bright reference light.

[0026] The calibration particles (multiple microparticles) 2001 will be described with reference to Figures 2(A) to (D). Figures 2(A) to (D) are schematic diagrams of the calibration particles 2001 in each example. As the material of the particles 2005 to which the fluorescent dye is bound, general particle materials can be used, for example, silica or polystyrene can be used. Since bio-derived materials have a low refractive index, silica is preferable as the material. The size of the calibration particles 2001 should preferably be equivalent to the size of the sample. When extracellular vesicles or viruses are assumed as the sample, the diameter of the particles 2005 should preferably be between 10 nm and 500 nm. More preferably, it should be between 40 nm and 200 nm.

[0027] The fluorescent dye 2002 for evaluation is used to calibrate the fluorescence measurement device 1000. It is desirable that the fluorescent dye 2002 for evaluation is the same dye used when measuring the sample. While the fluorescent dye 2002 and particle 2005 can be bound by common chemical reactions such as amide bonds or antibody reactions, it is desirable that the method of binding is the same as the method used to bind the sample to the fluorescent dye 2002. For example, if the fluorescent dye for evaluation is bound to the sample via an antibody, it is desirable that particle 2005 and the fluorescent dye 2002 for evaluation also be bound via an antibody. Similarly, if multiple antibodies are used to bind the sample to the fluorescent dye 2002, it is desirable that multiple antibodies are used to bind particle 2005 to the fluorescent dye 2002 for evaluation. If the target protein in the sample is a protein expressed on the sample membrane, it is desirable that the fluorescent dye 2002 for evaluation be bound to the surface of particle 2005. If the target protein is a protein expressed within the sample, it is desirable that the fluorescent dye 2002 used for evaluation be encapsulated within particle 2005.

[0028] The reference light (reference ray) is fluorescence from a fluorescent dye with different absorption and emission wavelengths than the evaluation fluorescent dye 2002. For example, the wavelength of the reference light is shorter than the wavelength of light from the fluorescent dye. This prevents the fluorescence emitted from the evaluation fluorescent dye from being absorbed by the reference fluorescent dye. However, the examples are not limited to this. Each example functions suitably by binding a fluorescent dye with such wavelength characteristics to the particle 2005 as the reference fluorescent dye 2006, as shown in Figure 2(A). Note that the method is not limited to fluorescent dyes; luminescent materials such as quantum dots may also be bound to the particles.

[0029] As shown in Figure 2(B), the reference fluorescent dye 2006 may be encapsulated within the particle 2005. By encapsulating the reference fluorescent dye 2006 within the particle 2005, it becomes possible to have more dye than if it were bound to the surface, and thus more reference fluorescence can be obtained. As shown in Figure 2(C), metal nanoparticles 2007 may be bound to the particle 2005. Since metal nanoparticles 2007 emit strong scattered light, the scattered light from the metal nanoparticles 2007 can be used as reference light. As shown in Figure 2(D), the scattered light 2008 emitted from particle 2005 may also be used as reference light. As will be described later, the reference light is used to determine the position and number of calibration particles, so any light that can achieve this can be used as reference light. In the following, the calibration particles shown in Figure 2(A) will be used as an example.

[0030] Referring to Figures 3 and 4, a method for evaluating the detection limit of the fluorescence measuring device 1000 with respect to the number of dyes using calibration particles 2001 will be explained. First, referring to Figure 3, a method for calculating the detection rate of calibration particles 2001 will be explained. Figure 3 is a schematic diagram showing the method for calculating the detection rate of calibration particles 2001.

[0031] A solution 2003 containing dispersed calibration particles 2001 is dropped onto a substrate 1400 to fix the calibration particles 2001 onto the substrate. The method of fixation is not particularly limited, but it is desirable that it be the same as the method used to fix the sample to the substrate. For example, if the sample and the substrate are chemically bound together with a ligand, it is desirable that the calibration particles be fixed to the substrate using a similar ligand. Also, if the solution containing the sample is fixed by drying, it is desirable that the calibration particles 2001 be fixed by drying in the same manner.

[0032] The control unit 1300 of the fluorescence measurement device 1000 acquires fluorescence images of fixed fluorescent particles. The fluorescence images consist of two images: a fluorescence image (first image) 2004 for the evaluation fluorescent dye 2002, and a fluorescence image (second image) 2009 for the reference fluorescent dye 2006 (reference light). The order of measurement is not limited, and measurements may be performed simultaneously.

[0033] Multiple bright spots, whose size is determined by the objective lens 1101, are observed in the image. Since both the evaluation fluorescence and the reference fluorescence are emitted from the same particle, the bright spots are located in approximately the same positions in the two fluorescence images 2004 and 2009. These bright spots are detected from the two images through image processing. The number of bright spots of the evaluation dye (the number of first bright spots included in fluorescence image 2004) N is obtained (extracted) from the evaluation fluorescence image 2004. In addition, the number of bright spots (the number of second bright spots included in fluorescence image 2009) N' is obtained (extracted) from the reference fluorescence image 2009.

[0034] The method for extracting bright spots is not particularly limited and can be performed using commonly used image processing methods. Since the reference dye is sufficiently bright, the number of bright spots in the reference fluorescence image 2009 indicates the number of particles present in that image. On the other hand, the bright spots in the evaluation fluorescence image 2004 indicate particles that emit fluorescence above the detection limit of the fluorescence measuring device 1000. Therefore, the proportion of particles detected (detection rate r) is calculated using the ratio of the number of bright spots N to the number of bright spots N', as shown in equation (1) below.

[0035]

number

[0036] Referring to Figure 4, we will explain how to calculate the detection limit relative to the number of dyes from the detection rate r. Figure 4 is a schematic diagram showing the method for calculating the detection limit.

[0037] In each embodiment, multiple types of solutions (particle dispersion solutions) in which calibration particles 2001 are dispersed are used. The multiple solutions (dispersion solutions) 20031, 20032, and 20033 differ in the number of evaluation fluorescent dyes 2002 bound to the calibration particles 2001 in each solution. In the solutions 20031, 20032, and 20033 shown in Figure 4, the number of evaluation fluorescent dyes 2002 increases in the order of the symbols. The control unit 1300 sets the detection rate r for each of the multiple solutions 20031, 20032, and 20033. i Obtain the following: i is the solution number, and the number of solutions is M, with values ​​from 1 to M. For convenience, the number of fluorescent dyes 2002 for evaluation increases in the order of the i numbers.

[0038] On the other hand, for each solution, the average number of fluorescent dyes 2002 bound per particle (number of dyes x i (Average value)￣x i (￣ is x i Measure the number of pigments (attached to the beginning of the character). Average number of pigments -x i This can be calculated from the absorbance and particle number concentration measurements of the solution. Since fluorescent particles are usually dispersed in the solution, the absorbance A of solution 2003 (20031-20033) can be measured. This can be done using a general spectrophotometer. Absorbance A is calculated using the molar extinction coefficient ε of the fluorescent dye and the concentration c of the dye. A Using the thickness L of the container holding the solution, it can be expressed as shown in equation (2) below.

[0039]

number

[0040] The molar absorption coefficient ε is generally known. Therefore, using Equation (2), the concentration c of the dye A is obtained by the following Equation (3).

[0041]

Equation

[0042] In addition to measuring the absorbance, the number of particles (particle number concentration) c in the unit volume of the solution 2003 containing the calibration particles 2001 is measured. This can be measured by a general dynamic light scattering method or nanoparticle tracking analysis method. Alternatively, it can also be obtained by comparing the absorption spectrum of a solution with a known concentration and the absorption spectrum of the solution 2003 containing the calibration particles. p By taking the ratio of the previously obtained c A and c p the average number of dyes ￣x can be known from the following Equation (4).

[0044]

Equation

[0045] If this measurement is performed for all solutions, the average number of dyes ￣x i for each solution can be known.

[0046] The lower graph in Figure 4 shows the evaluation results based on the obtained dataset of the average number of dyes ￣x i and a plurality of detection rates r i In this graph, the horizontal axis represents the average number of dyes ￣x, and the vertical axis represents the detection rate r respectively. As shown in Figure 4, as the average number of dyes ￣x increases, the detection rate r increases.

[0047] The control unit 1300 uses the dataset (￣x i , r i ​​​​​​​The detection limit of the fluorescence measuring device 1000 for the number of dyes is estimated from the following. Particles dispersed in a solution have a randomly different number of dyes bound to each particle. When the number of fluorescent dyes bound to one particle is x, the probability of a particle with x fluorescent dyes appearing in the i-th solution is given by the probability density function f i It is thought to follow (x). Detection rate r i The number of dyes x is the detection limit of the fluorescence measuring device 1000 (the minimum number of fluorescent dyes that can be detected by the fluorescence measuring device 1000) x min Since this is the proportion of particles greater than or equal to f i The cumulative distribution function F of (x) i Using (x), we obtain the following relationship (5).

[0048]

number

[0049] The number of dyes that bind is random and non-negative, so the probability density function f i A good example of (x) is the log-normal distribution function, namely, f i (x) can be expressed as shown in equation (6) below.

[0050]

number

[0051] μ i σ and σ are coefficients that determine the log-normal distribution function (detection rate r), respectively. i This is the coefficient associated with the distribution. Due to the characteristics of the log-normal distribution, σ is a quantity corresponding to the ratio of the mean to the standard deviation, and is considered to be the same between solutions in the range where the pigment concentration does not change significantly. On the other hand, μ is the mean of the log-normal distribution, i.e., the average number of pigments bound to the pigment ￣x. i This corresponds to a different value between solutions, and therefore the amount will differ between solutions.

[0052] By using the cumulative distribution function for a log-normal distribution, equation (5) can be expressed as equation (7) below.

[0053]

number

[0054] erf is the error function.

[0055] Due to the properties of the log-normal distribution, the average number of pigments is ￣x. i The coefficient μ i Using σ, it can be expressed as shown in equation (8) below.

[0056]

number

[0057] Equation (8) is the coefficient μ i Solving for and substituting into equation (7), we obtain the following equation (9).

[0058]

number

[0059] Equation (9) is given by the dataset (￣x i ,r i This is a function that represents the relationship between (￣x). Using this function, the dataset (￣x) as the measurement result is generated. i ,r i ) best reproduces x min By finding σ through fitting, etc., x min This allows us to understand the following. For example, in Figure 4, we can obtain fitting results like the solid line in the graph.

[0060] Next, with reference to Figure 5, the calibration method (measurement method) of the fluorescence measuring device 1000 in each embodiment will be described. Figure 5 is a flowchart of the measurement method.

[0061] First, in step S1, the control unit 1300 of the fluorescence measuring device 1000 acquires fluorescence images using calibration particles (measuring fluorescence using multiple calibration particles). That is, the calibration particles are fixed on the substrate 1400, and the control unit 1300 acquires fluorescence images 2004 and 2009 using light sources and fluorescence filters with wavelengths corresponding to each fluorescent dye.

[0062] Next, in step S2, the control unit 1300 performs image processing on the acquired fluorescence images 2004 and 2009, and determines the number of bright spots N in each fluorescence image. i , N i Obtain (calculate) ' and use formula (1) to obtain the detection rate r i Obtain (calculate) it.

[0063] Next, in step S3, the control unit 1300 determines the detection rate r i , and the average number of pigments measured beforehand ￣x i Using a probability density function that includes the detection limit x for the number of dyes in the fluorescence measurement device 1000, min The average number of pigments is obtained (calculated) using equation (9). For example, the control unit 1300 uses equation (9) to obtain (calculate) the average number of pigments ￣x i and detection rate r i The dataset that best reproduces x min σ is calculated by fitting.

[0064] Next, in step S4, the control unit 1300 sets the detection limit x for the number of dyes recorded in the fluorescence measuring device 1000. min The value is changed (updated) to the value obtained in steps S1 to S4. The value can be changed by the user inputting the calculation result into the fluorescence measuring device 1000 via an input device, or the fluorescence measuring device 1000 may update it automatically.

[0065] Steps S1 to S4 are calibration steps for calibrating the fluorescence measuring device 100. Since the sensitivity of the fluorescence measuring device 1000 changes due to aging and changes in the external environment, it is desirable to perform the calibration steps periodically, and preferably after each measurement.

[0066] Next, in step S5, the control unit 1300 measures the fluorescence of the sample. Specifically, the control unit 1300 fixes the sample, which has been labeled with a fluorescent dye for evaluation, onto the substrate 1400 and acquires a fluorescence image.

[0067] Next, in step S6, the control unit 1300 performs image processing on the fluorescence image obtained in step S5 to obtain (calculate) the number of bright spots in the fluorescence image, i.e., the number of detected spots.

[0068] Using the method described above, the detection limit x of the fluorescence measuring device 1000 relative to the number of dyes can be determined. min It can measure with high precision. The detection limit x over time min By evaluating this, it is possible to understand not only the deterioration status and maintenance timing of the fluorescence measuring device 100, but also the detection limit x min By comparing fluorescence measurement devices, it is possible to evaluate the superiority or inferiority of each device. Furthermore, the detection limit for the number of dyes is defined as the number of dyes in a single sample that exceeds the detection limit x. min This means that if the sample has the above number of dyes, it can be detected from fluorescence image 2004. Knowing the detection limit for the number of dyes means that the sample detected by fluorescence measuring device 1000 will have at least the detection limit x min We can guarantee that the above-mentioned fluorescent dyes are present.

[0069] Furthermore, the number of known dyes β per ligand of a ligand such as an antibody used to bind a dye to a sample can be used. By using the number of dyes β, the minimum amount of protein per sample required for detection by the fluorescence analyzer 1000 (the minimum measurable amount), i.e., the detection limit y for the amount of protein, can be expressed by the following equation (10): min It is possible to obtain (calculate) this.

[0070]

number

[0071] To put it another way, the detection limit for protein mass is the detection limit y for protein mass in a single sample.min This means that if the above proteins are expressed, this sample can be detected from fluorescence image 2004. The detection limit y is determined before measuring the sample. min By evaluating this, the detected sample will have at least a detection limit y min We can guarantee that the above proteins are being expressed.

[0072] In each example, the value calculated and updated in the calibration step is the detection limit x relative to the number of dyes. min It is not limited to the detection limit y for the number of proteins. min But that's fine.

[0073] Next, with reference to Figure 6, a calibration method (measurement method) as a modified example of each embodiment will be described. Figure 6 is a schematic diagram showing the measurement method as this modified example. As shown in Figure 6, when detecting bright spots from fluorescence image 2004, two fluorescence images 2004 and 2009 may be compared to identify bright spots occurring at approximately the same location.

[0074] Because the light from the evaluation dye is weak, unexpected impurities may emit light or noise generated by the image sensor may be mixed into the fluorescence image 2004. Since it is rare for such impurities or noise to be observed at approximately the same location in the two images, it is possible to remove unwanted signals by identifying only the bright spots that occur in the same location in the two images. Here, "approximately the same location" does not mean exactly the same location, but rather a range within which the coordinates coincide, for example, several times the size of the point image distribution function of the objective lens 1101, or within a few pixels. By calculating the number N of bright spots identified as being at the same location and calculating the detection limit using the method described above, high-precision calibration with reduced influence of unwanted signals becomes possible.

[0075] In each embodiment, the log-normal distribution function was described as the probability density function, but it is not limited to this. Functions similar to the log-normal distribution, such as the gamma distribution, beta distribution, and Weibull distribution, are known and may be used as the probability density function f(x) for analysis. In any of these functions, multiple coefficients representing the probability density function are related by the detection rate expressed in equation (5) and the average number of pigments corresponding to equation (8). Based on these relationships, the detection limit x of the fluorescence measurement device 1000 can be determined by analyzing the dataset of detection rate and average number of pigments through fitting, etc. min It is possible to calculate the following. The log-normal distribution is: number of pigments x i Because it accurately represents the frequency of an event and is easy to handle, it is a desirable probability density function.

[0076] In each embodiment, the method for calculating the coefficient of the probability density function was described as being obtained by fitting, but this is not the only method. σ in a log-normal distribution is a value that is generally determined by the particle. By performing the above method using a fluorescence measuring device other than the fluorescence measuring device 1000 to be evaluated and obtaining σ, the detection limit x can be determined without using fitting by solving equation (9). min It is possible to find this.

[0077] Each example can also be used to evaluate calibration particles. Different calibration particles are measured using the same fluorescence measuring device 1000, and the detection limit x min By comparing the values ​​of each particle, we can evaluate their superiority or inferiority.

[0078] The following describes each embodiment in detail.

[0079] (Example 1) First, let's describe Example 1. The fluorescence measuring device 1000 of this embodiment, shown in Figure 1, has two light sources 1201: an LED with a central wavelength of 475 nm and an LED with a central wavelength of 630 nm.

[0080] As filter cubes (fluorescence filters) 1204, fluorescence filter set 1 for observing green fluorescent dyes and fluorescence filter set 2 for observing red fluorescent dyes are provided in the turret. Fluorescence filter set 1 has an excitation filter with a center wavelength of 480 nm and a bandwidth of 30 nm, a long-pass dichroic mirror with a cut-on wavelength of 505 nm, and an absorption filter with a center wavelength of 535 nm and a bandwidth of 40 nm. Fluorescence filter set 2 has an excitation filter with a center wavelength of 620 nm and a bandwidth of 50 nm, a long-pass dichroic mirror with a cut-on wavelength of 655 nm, and an absorption filter with a center wavelength of 690 nm and a bandwidth of 50 nm.

[0081] The illumination unit 1200 has a general collimator lens 1202 and a focusing lens 1203, and together with the objective lens 1101, it constitutes a Köhler illumination system. The objective lens 1101 has a magnification of 40x and an NA of 0.95. The fluorescence image of the sample 1500 fixed on the substrate 1400 is formed on the image sensor (CMOS sensor) 1103 via the objective lens 1101, the filter cube 1204, and the imaging lens 1102.

[0082] Calibration particles are created by binding silica particles with a cyanine-based red fluorescent dye having an NHS Ester at its end as the evaluation fluorescent dye, and a fluorescein-based green fluorescent dye having an NHS Ester at its end as the reference fluorescent dye. The silica particles have a diameter of approximately 100 nm and have amino groups on their surface. A sufficient amount of the reference green fluorescent dye is bound to the silica particles. Five types of calibration particles are created, each with a different amount of the evaluation red fluorescent dye bound to it. These particle dispersions are dropped onto a glass substrate and a Si substrate with gold deposited on its surface, and the calibration particles are fixed by drying. Fluorescence images of the fixed particles are acquired using light source wavelengths and fluorescence filters corresponding to the two fluorescent dyes.

[0083] By performing image processing on the obtained fluorescence images, bright spots are extracted, and the detection rate r for each solution is determined. i(i=1~5) is calculated. Additionally, the absorbance and particle number concentration are measured for each particle solution to determine the average number of pigments in each solution. i Calculate (i=1~5).

[0084] Figure 7 shows the obtained detection rate r i This figure shows a dataset of the average number of pigments ￣x. In Figure 7, the horizontal axis represents the average number of pigments ￣x, and the vertical axis represents the detection rate r. Furthermore, a log-normal distribution is assumed as the probability density function, and the detection rate r i and the relationship between the average number of pigments and x best reproduces x min σ is calculated by fitting based on equation (9). The obtained x min The result was 561 when glass was used, and 53 when a gold-deposited substrate was used. As described above, x min This allows us to determine the detection limit. Furthermore, a gold-deposited substrate has a lower detection limit than a glass substrate. Thus, it is possible not only to determine the detection limit, but also to compare substrates and devices.

[0085] Furthermore, assuming a typical number of pigments β bound per ligand is 5, then from equation (10), the detection limit y for the amount of protein in the fluorescence measurement device 1000 is... min The value is calculated as 112 when using a glass substrate and 11 when using a gold-deposited substrate.

[0086] (Example 2) Next, Example 2 will be described. In this example, a flow cytometer 3000 is used as the fluorescence measurement device. Figure 8 is a schematic diagram of the flow cytometer (measurement device) 3000 in this example.

[0087] The flow cytometer 3000 irradiates calibration particles 2001 flowing through channel 3001 with light from laser sources 3002 and 3003. The fluorescence emitted from the calibration particles 2001 is detected by photomultiplier tubes 3006 and 3007 after passing through bandpass filters 3004 and 3005. Channel 3001 has a narrow channel, such that only one particle can flow through it in the laser irradiation area. Laser source 3002 is a semiconductor laser with a wavelength of 488 nm. Laser source 3003 is a semiconductor laser with a wavelength of 635 nm. Bandpass filter 3004 has a center wavelength of 525 nm and a bandwidth of 50 nm. Bandpass filter 3005 has a center wavelength of 700 nm and a bandwidth of 50 nm.

[0088] The calibration particles are the same as those used in Example 1. The calibration particles are flowed at a flow rate sufficient to determine that the fluorescence of the evaluation dye and the signal of the reference dye were emitted from the same particle at the same time, and the fluorescence intensity of each is measured. In this example, the current value output from photomultiplier tubes 3006 and 3007 is the fluorescence intensity. Let N' be the number of reference fluorescence observed (second bright spot count), and N be the number of evaluation fluorescence measured (first bright spot count). Based on equation (1), the detection rate r for each particle is calculated. i Measure the detection rate r. i Furthermore, the detection limit can be determined by performing the same calculation as in Example 1 using a dataset with an average number of pigments ￣x.

[0089] The flow cytometer 3000 may have a photomultiplier tube for measuring side scattering. When the particles shown in Figure 2(C) or Figure 2(D) are used as calibration particles 2001, the side-scattered light can be used as reference light. The wavelength characteristics of the laser light sources 3002 and 3003, and the bandpass filters 3004 and 3005 are appropriately changed depending on the fluorescent dye being evaluated.

[0090] (Other examples) The present invention can also be realized by supplying a program that implements one or more of the functions of the above-described embodiments to a system or device via a network or storage medium, and by having one or more processors in the computer of that system or device read and execute the program. It can also be realized by a circuit (e.g., an ASIC) that implements one or more functions.

[0091] Each embodiment's disclosure includes the following configuration and method. (Method 1) A step of measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the plurality of fine particles, using a plurality of solutions in which a plurality of fine particles labeled with a fluorescent dye and emitting a reference light are dispersed, A step of obtaining, with respect to each of the plurality of solutions, the proportion (r) of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measurement method characterized by comprising the step of obtaining the minimum number of fluorescent dyes detectable by a measuring device based on the respective proportions of the plurality of solutions. (Method 2) The measurement method according to Method 1, characterized in that the average number of fluorescent dyes bound to the fine particles differs from one another with respect to the plurality of solutions. (Method 3) The measurement method according to method 1 or 2, characterized in that, in the step of obtaining the minimum number, the minimum number is obtained using a function that includes the ratio. (Method 4) The measurement method according to method 3, characterized in that the function is a log-normal distribution function having coefficients associated with the proportion. (Method 5) The proportion of the i-th solution among the plurality of solutions is r i , the minimum number is x min The cumulative distribution function of the probability density function of the presence of the microparticles to which the number of fluorescent dyes x are bound in the i-th solution is F. i When (x) is given, the above function is JPEG2026092804000012.jpg1062

[0092] The measurement method according to method 3 or 4, characterized in that it is expressed by the formula shown. (Method 6) The measurement method according to any one of methods 1 to 5, further comprising the step of updating the minimum number held in the measuring device using the minimum number. (Method 7) In the step of obtaining the aforementioned ratio, The plurality of microparticles are fixed on a substrate, and a first image of the plurality of microparticles with respect to the fluorescent dye and a second image with respect to the reference light are obtained. The first number of bright spots located at the same position in the first and second images, and the second number of bright spots included in the second image are obtained. A measurement method according to any one of methods 1 to 6, characterized in that the ratio of the particles is obtained by obtaining the ratio of the first number of bright spots to the second number of bright spots. (Method 8) The measurement method according to any one of methods 1 to 7, further comprising the step of measuring the fluorescence of a sample labeled with the same fluorescent dye as the aforementioned fluorescent dye. (Method 9) The measurement method according to any one of methods 1 to 8, characterized in that the diameter of the fine particles is from 10 nm to 500 nm. (Method 10) The measurement method according to any one of methods 1 to 9, characterized in that the fine particles are silica or polystyrene. (Method 11) The measurement method according to any one of methods 1 to 10, characterized in that the reference light is scattered light from the fine particles or fluorescence from a fluorescent dye different from the fluorescent dye labeled on the fine particles. (Method 12) The measurement method according to any one of methods 1 to 11, further comprising the step of obtaining the minimum amount of protein that can be measured by the measuring device using the aforementioned minimum number. (Method 13) A measurement method according to any one of methods 1 to 12, further comprising the step of obtaining the number of samples detected. (Method 14) The measurement method according to any one of methods 1 to 13, characterized in that the wavelength of the reference light is shorter than the wavelength of the light from the fluorescent dye. (Method 15) The measurement method according to any one of methods 1 to 14, characterized in that the fine particles are extracellular vesicles or viruses. (Method 16) The measurement method according to any one of methods 1 to 15, characterized in that, in the measurement step, the minute particles are fixed on a plasmon substrate. (Composition 1) A measuring means for measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the multiple microparticles, using multiple solutions in which multiple microparticles labeled with a fluorescent dye for evaluation and emitting reference light are dispersed, A first acquisition means for each of the plurality of solutions to acquire the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measuring device characterized by having a second acquisition means for acquiring the minimum number of fluorescent dyes detectable by the measuring device based on the respective proportions of the plurality of solutions. (Configuration 2) A program characterized by causing a computer to execute any of the measurement methods described in Methods 1 to 6. (Composition 3) A computer-readable storage medium characterized by storing the program described in Configuration 2.

[0093] Although preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and various modifications and changes are possible within the scope of its essence. [Explanation of Symbols]

[0094] 1000 Fluorescence measuring device (measuring device) 2001 Calibration particles (microparticles) 2002 Fluorescent dyes 3000 Flow cytometer (measuring device)

Claims

1. A step of measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the plurality of fine particles, using a plurality of solutions in which a plurality of fine particles labeled with a fluorescent dye and emitting a reference light are dispersed, A step of obtaining, with respect to each of the plurality of solutions, the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measurement method characterized by comprising the step of obtaining the minimum number of fluorescent dyes detectable by a measuring device based on the respective proportions of the plurality of solutions.

2. The measurement method according to claim 1, characterized in that the average number of fluorescent dyes bound to the fine particles differs from one another with respect to the plurality of solutions.

3. The measurement method according to claim 1, characterized in that, in the step of obtaining the minimum number, the minimum number is obtained using a function that includes the ratio.

4. The measurement method according to claim 3, characterized in that the function is a log-normal distribution function having coefficients associated with the proportion.

5. The proportion of the i-th solution among the plurality of solutions is r ｉ , the minimum number is x ｍｉｎ The cumulative distribution function of the probability density function of the minute particles to which the number x of the fluorescent dyes are bound is F. ｉ When (x) is used, the above function is, The measurement method according to claim 3, characterized in that it is expressed by the formula.

6. The measurement method according to claim 1, further comprising the step of updating the minimum number held in the measuring device using the minimum number.

7. In the step of obtaining the aforementioned ratio, The plurality of microparticles are fixed onto a substrate, and a first image of the plurality of microparticles with respect to the fluorescent dye and a second image with respect to the reference light are obtained. The first number of bright spots located at the same position in the first and second images, and the second number of bright spots included in the second image are obtained. The measurement method according to any one of claims 1 to 6, characterized in that the ratio of the particles is obtained by obtaining the ratio of the first number of bright spots to the second number of bright spots.

8. The measurement method according to any one of claims 1 to 6, further comprising the step of measuring the fluorescence of a sample labeled with the same fluorescent dye as the aforementioned fluorescent dye.

9. The measurement method according to any one of claims 1 to 6, characterized in that the diameter of the fine particles is from 10 nm to 500 nm.

10. The measurement method according to any one of claims 1 to 6, characterized in that the fine particles are silica or polystyrene.

11. The measurement method according to any one of claims 1 to 6, characterized in that the reference light is scattered light from the minute particles or fluorescence from a fluorescent dye different from the fluorescent dye labeled on the minute particles.

12. The measurement method according to any one of claims 1 to 6, further comprising the step of obtaining the minimum amount of protein that can be measured by the measuring device using the aforementioned minimum number.

13. The measurement method according to any one of claims 1 to 6, further comprising the step of obtaining the number of samples detected.

14. The measurement method according to any one of claims 1 to 6, characterized in that the wavelength of the reference light is shorter than the wavelength of the light from the fluorescent dye.

15. The measurement method according to any one of claims 1 to 6, characterized in that the minute particles are extracellular vesicles or viruses.

16. The measurement method according to any one of claims 1 to 6, characterized in that, in the measurement step, the minute particles are fixed on a plasmon substrate.

17. A measuring means for measuring the fluorescence of the fluorescent dye and the reference light emitted from individual particles among the plurality of fine particles, using a plurality of solutions in which a plurality of fine particles labeled with a fluorescent dye and emitting a reference light are dispersed, A first acquisition means for each of the plurality of solutions to acquire the proportion of particles among the plurality of fine particles in which both the fluorescence of the fluorescent dye and the reference light are detected, A measuring device characterized by having a second acquisition means for acquiring the minimum number of fluorescent dyes detectable by the measuring device based on the respective proportions of the plurality of solutions.

18. A program characterized by causing a computer to execute the measurement method described in any one of claims 1 to 6.

19. A computer-readable storage medium characterized by storing the program described in claim 18.

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